Biological system for degrading nitroaromatics in water and soils

ABSTRACT

Novel methods for biodegrading nitroaromatic compounds present as contaminants in soil or water using microorganisms are disclosed. Water is treatable directly; dry soil is first converted into a fluid medium by addition of water. The preferred method comprises two stages, each employing microorganisms: a fermentative stage, followed by an anaerobic stage. The fermentative stage is rapid, wherein an inoculum of aerobic and/or facultative microorganisms ferments a carbohydrate added to the fluid medium, exhausting the oxygen in the fluid medium and thereby inhibiting oxidative polymerization of amino by-products of the nitroaromatics. In the subsequent anaerobic stage, an inoculum of a mixed population of anaerobic microorganisms completes the mineralization of the contaminant nitroaromatics, using the remaining carbohydrate as a carbon and energy source. Preferably, the carbohydrate is a starch and the aerobic and/or facultative microorganisms are amylolytic, which cleave the starch at a moderate rate throughout both stages, ensuring a sustained supply of metabolizable carbohydrate. The microorganisms are preferably selected to be resistant to the types and concentrations of nitroaromatics present as contaminants.

This is a continuation of application Ser. No. 08/229,462, filed Apr.18, 1994, now abandoned, which is a continuation of Ser. No. 08/096,735,filed Jul. 23, 1993, now issued U.S. Pat. No. 5,387,271, which is a filewrapper continuation-in-part of Ser. No. 07/508,056, filed Apr. 11,1990, now abandoned.

FIELD OF THE INVENTION

This invention relates to the biodegradation of various nitroaromaticcompounds in water and soils, including dinoseb(2-(1-methylpropyl)-4,6-dinitrophenol), using microorganisms.

BACKGROUND OF THE INVENTION

Certain nitrophenolic compounds are sufficiently toxic to life to renderthem effective for use as herbicides, insecticides, or miticides. Suchcompounds include dinoseb (2-(1-methylpropyl)-4,6-dinitrophenol) whichhas been widely used as a herbicide since the 1950's on a variety ofcrops in the United States. Concerns for the safety of agriculturalworkers has resulted in discontinued use of dinoseb. However, numeroussites remain contaminated with this compound.

Other nitroaromatic compounds are similar to dinoseb in terms ofchemical structure, but have other applications, such as in explosives.Such compounds include trinitrotoluene (TNT) and dinitrotoluene (DNT).Because of the widespread use of these compounds over a lengthy periodof time, many sites have become contaminated with these compounds,including both manufacturing and military sites.

Many nitroaromatic compounds are either poorly degradable ornondegradable in field environments outside the laboratory. Previously,land farming was the favored method for disposing of these and otherchemicals, wherein the chemicals were mixed with soil, fertilizer wasadded, and the mixture aerated to promote microbial activity.Unfortunately, nitroaromatics were not satisfactorily degraded by landfarming or other well-aerated processes. Possible reasons include lackof nitroaromatic-degrading microorganisms, partitioning of thecontaminant chemicals to biologically sequestered or inhospitable partsof the environment, and accumulation of toxic partial-breakdownby-products. Problems with land farming in general included the slowrate of biodegradation, high expense, and accumulation of toxicby-products.

Other methods have been used to remove nitroaromatics and similarcompounds from contaminated soils, but with little practical success.Such methods include transportation of contaminated soil to hazardouswaste dumps, and on-site incineration of the soil. Problems with suchmethods include high cost and poor accountability of the responsibleparty.

Previous laboratory studies indicated that certain nitroaromaticmolecules are susceptible at least to microbiological transformation.However, the studies did not disclose biochemical mechanisms of suchtransformation or degradation or whether the nitroaromatic compoundswere completely mineralized. In one study, for example, a soil Moraxellamicroorganism was isolated that was capable of growth on p-nitrophenolas its only source of carbon and energy. Spain et al., Biochem. Biophys.Res. Comm. 88:634-641 (1959). In another study, the anaerobic bacteriumVeillonella alkalescens reductively transformed nitroaromatic compounds,converting the nitro groups to amino groups. McCormack et al., Appl.Environ. Microbiol. 31:949-958 (1976).

Aminoaromatic derivatives of nitroaromatics can undergo enzymaticoxidation to form polymeric (large molecular weight) materials. Parris,Residue Revs. 76:1-30 (1980). In the field, such polymers are usuallyincorporated into soil humic matter. Channon et al., Biochem. J.38:70-85 (1944); McCormick. et al., Appl. Environ. Microbiol. 31:949-958(1976); Simmons et al., Environ. Sci. Technol. 23.:115-121 (1989). Humicmatter tends to be long-lived in soils, thereby representing a majorlong-term environmental fate of many nitroaromatics and aminoaromatics.Other soil microorganisms are capable of cleaving the azo linkages ofpolymerized aminoaromatics, often forming toxic by-products.

Bacteria are also able to attack nitrobenzoic acid, Cartwright and Cain,Biochem. J. 71:248-261 (1959), as well as o-nitrophenol andm-nitrophenol, Zeyer and Kearney, J. Agric. Food Chem. 32:238-242(1984), where the nitro group is released as nitrite. Again, however,complete mineralization has not been demonstrated. Further, nitriterelease has not been found to be a significant pathway for highlysubstituted nitroaromatics. No instance is currently known where acompound possessing more than one nitro substituent has been completelymineralized. In fact, the pertinent literature presents no evidencesupporting ring cleavage of highly substituted nitroaromatics. Kaplan,"Biotransformation Pathways of Hazardous Energetic Organo-NitroCompounds," in Biotechnology and Degradation. Adv. Appl. Biotechnol.Ser. 4:155-181, Gulf Pub. Co., Houston, Tex. (1990).

Aromatic groups in general appear to be degradable via only a fewaerobic and anaerobic pathways. Gottschalk, Bacterial Metabolism, 2ded., Springer Verlag, New York (1986), pp. 157-162; Berry et al.,Microbiol. Rev. 51:43-59 (1987); Schink, "Principles and Limits ofAnaerobic Degradation: Environmental and Technological Aspects," inZinder (ed.), Biology of Anaerobic Microorganisms, Wiley, N.Y. (1988).Aerobically, many aromatic groups are degraded to catechol,protocatechuate or homogentisate by the action of oxygenase anddioxygenase enzymes. Catechol and protocatechuate can be degradedfurther by aromatic ring cleavage either ortho or meta to the hydroxylgroups. Because of the difficulty of working with anaerobicmicroorganisms and processes, biochemical pathways describing anaerobicdegradation of aromatic compounds have been less well characterized.

Alkyl groups on aromatic rings are degradable via reactions similar tothose for simple alkanes. Under aerobic conditions, the terminal carbonis oxidized to yield a carboxylic acid. Degradation then proceeds byβ-cleavage to yield either benzoates (odd-numbered carbon chains) orphenylacetates (even-numbered carbon chains). No anaerobicmicroorganisms capable of carrying out this process have been isolatedto date. In spite of the above results known in the art, there is littleinformation currently available on practical means of using microbialcultures to bioremediate nitroaromatic-contaminated soils.

Dinoseb, an intensely yellow-colored compound visible at concentrationsas low as 10 ppm, has been found to not significantly accumulate inagricultural soil at normal application rates, even after years ofrepeated application. Doyle et al., J. Agric. Food Chem. 26:987-989(1978). However, higher application rates, such as from spills ofsubstantial amounts of the compound, can result in appreciableaccumulation at a site. Presumably, therefore, dinoseb at lowerconcentrations is transformed by certain soil microorganisms. Suchtransformation appears to result only in the formation of amino andacetoamido forms of dinoseb, which apparently retain significanttoxicity. Parris, Residue Revs. 76:1-30 (1980).

Previous work on the biotransformation of the explosive2,4,6-trinitrotoluene (TNT) indicates that the primary mode involvestransformation (reduction) of the nitro groups. Kaplan, supra. A recentpaper from Soviet researchers describes degradation of TNT by a strainof Pseudomonas fluorescens. Naumova et al., Mikrobiologiya 57:218(1988). But, while these reports shed some light on microbial events andhypothetical biochemical mechanisms therefor, they neither disclose norsuggest effective methods for bioremediating soils or wastewatercontaminated with these compounds. Further, the Soviet results have notbeen confirmed outside the U.S.S.R.

Hence, although several anaerobic microbiological systems have beendescribed for degrading other aromatic chemicals, little to noinformation is available on practical means of using these cultures tobioremediate contaminated soils and waters, especially soils and waterscontaminated with nitroaromatics. In today's world, effectiveremediation of environmental sites contaminated with compounds, such asnitroaromatics, requires that the contaminants be completely mineralizedto ensure the. absence of latently toxic by-products. Such results fornitroaromatics simply have not been shown in the prior art, particularlyas applicable to large-scale, low-cost bioremediation efforts.

Therefore, there remains a need for a method to effectively bioremediatedinoseb-contaminated soils, as well as soils contaminated with othernitroaromatic compounds, such as TNT and DNT.

Further, there is a need for such a method that can be performed at anatural site contaminated with dinoseb or a related nitroaromaticcompound.

Further, there is a need for such a method that can completely degradedinoseb and other nitroaromatics, leaving no detectable orenvironmentally significant amounts of aromatic by-products or othertoxic intermediary compounds, including polymeric derivatives.

Further, there is a need for such a method employing microorganisms oftypes and species profiles normally found in many soil environments.

Further, there is a need for such a method that is inexpensive and easyto perform, particularly on a large-scale, in the field.

Further, there is a need for such a method that can be performedrapidly, including in the field.

Further, there is a need for such a method that will effectbioremediation of nitroaromatic-contaminated soil without specializedbioreactors or other complex equipment.

SUMMARY OF THE INVENTION

In accordance with the present invention, soil or water contaminatedwith one or more nitroaromatic compounds is subjected to a two-stagebioremediation process employing different microorganisms during eachstage. The stages comprise an initial fermentation stage followed by ananaerobic stage. Most of the actual biodegradation of the contaminantnitroaromatics takes place in the anaerobic stage. At the end of theanaerobic stage, the contaminant nitroaromatics have been biodegraded tonon-toxic end-products.

As another aspect of the invention, complete biodegradation ofnitroaromatics has been found to occur only under anaerobic conditions.Since anaerobiosis generally requires an aqueous environment, it isusually necessary to add extraneous water to anitroaromatic-contaminated soil to create a fluid mud slurry of the soilbefore beginning the process. Contaminated water can be subjected to theprocess directly.

During the first stage of the process, the normally aerobic contaminatedsoil (with water added to form a mud slurry) or contaminated water aloneis rendered anaerobic. The preferred method for achieving an anaerobiccondition is by a fermentation of a supply of starchy carbohydrate orother readily fermentable carbon source added to the slurry or water.Fermentation, where the carbon source is a starchy carbohydrate, isperformed by one or more species of aerobic or facultatively anaerobicamylolytic microorganisms inoculated into the slurry or water.Amylolytic microorganisms are not required if the carbon source is asimple sugar, such as fructose or glucose.

As another aspect of the invention, the aerobic or facultativemicroorganisms are preferably isolated and enriched in a culturecontaining the particular nitroaromatic present in the contaminated soilor water.

As another aspect of the invention, it is usually necessary at thebeginning of the aerobic stage to add an extraneous source of nitrogenfor the microorganisms. The nitrogen source is preferably in the form ofammonium ion or simple amino compounds readily utilizable by aerobic andanaerobic microorganisms.

As another aspect of the invention, it is preferable to stimulate arapid onset of intense fermentation to quickly cause exhaustion of theoxygen dissolved in the slurry or water, thereby rendering the slurry orwater anaerobic, without exhausting the carbon source. Quick attainmentof anaerobiosis minimizes oxidative polymerization of any aminoderivatives of the nitroaromatics that tend to form under aerobicconditions. Once formed, such polymers are difficult to biodegrade.Rapid anaerobiosis can be achieved by inoculating the slurry or waterwith a large dosage of aerobic and/or facultative fermentativemicroorganisms.

If necessary, mineral nutrients, including phosphate salts, can be addedto the soil slurry or water to facilitate microbial growth.Supplementary vitamins and cofactors may also be required, but probablyonly when treating wastewaters having very little dissolved organiccarbon. Soils generally have sufficient nutrients.

As another aspect of the invention, the carbon source added to the soilslurry or water for aerobic fermentation is preferably a starchycarbohydrate substance hydrolyzable to constituent sugars by amylolyticmicroorganisms in the aerobic inoculum. A starch is preferred overmerely adding free sugar because the starch serves as a reservoir ofmetabolizable carbohydrate that ensures an adequate supply of easilymetabolizable sugar for the microorganisms, both in the aerobic stageand in the anaerobic stage. Anaerobic biodegradation of nitroaromaticsrequires such a sustained sugar supply, however, too high sugarconcentrations inhibit dinoseb degradation. A supply of sugar added atthe beginning of the aerobic stage would generally be exhaustedprematurely, making it difficult to maintain anaerobic conditions forthe requisite amount of time to achieve complete mineralization of thenitroaromatics.

As another aspect of the invention, the amount of starchy carbohydrateto be added to the volume of soil slurry or water to be treated can be"tailored" to ensure the desired degree of biodegradation is attainedand waste of carbohydrate is avoided. The amount of carbohydrate shouldbe just sufficient to supply the metabolic needs of the microorganismsuntil the anaerobic biodegradation of contaminant nitroaromatics iscomplete.

As yet another aspect of the invention, once strict anaerobic conditionshave been attained in the volume of slurry or water, an inoculumcomprised of an anaerobic consortium of microorganisms is added to thevolume to start the second, or anaerobic, stage. Anaerobic conditionsare preferably determined via a potentiometric measurement, where aredox potential of -200 mV or less indicates strict anaerobicconditions.

As yet another aspect of the invention, the anaerobic consortiumcomprises multiple species of microorganisms that have been grown in amedium containing one or more nitroaromatics identical or similar to thecontaminant nitroaromatics to be biodegraded. The anaerobicmicroorganisms are able to biodegrade the nitroaromatics in the presenceof metabolizable sugar to simple non-toxic compounds, such as methane,carbon dioxide, and acetate.

As yet another aspect of the invention, after inoculation, the anaerobicconsortium is afforded sufficient time to biodegrade the contaminantnitroaromatics in the soil slurry or water to non-toxic end-products.Because degradation of the nitroaromatic compounds occurs in ananaerobic environment, polymerization to toxic humic-like compounds andother large, long-lived, latently toxic molecules normally formed inaerobic environments is prevented.

As yet another aspect of the invention, the present method is preferablyperformed in a suitably large covered vessel for containing thecontaminated soil slurry or water during bioremediation. Suchcontainment ensures that anaerobic conditions in the soil slurry orwater are reached more rapidly and are better controlled and maintained.Containment also facilitates easier control of environmental parameters,such as temperature, pH, and, if needed, escape of volatile gases fromthe slurry or water being treated.

It is accordingly one object of the present invention to provide amethod for effectively bioremediating soils and waters contaminated withone or more nitroaromatic compounds.

Another object of the present invention is to provide such a method thatcan be performed at natural sites contaminated with nitroaromatics.

Another object of the present invention is to provide such a method thatwill allow contaminant nitroaromatics in soil or water to be biodegradedto such an extent that no detectable or environmentally significantamounts of aromatic by-products or other toxic intermediary compoundsare left in the soil or water, including latently toxic polymericderivatives of the nitroaromatics.

Another object of the present invention is to provide such a method thatutilizes microorganisms similar to those found in many soil and aquaticenvironments.

Another object is to provide such a method that is inexpensive and easyto perform, particularly on a large scale in the field.

Another object is to provide such a method that can be performedrapidly, even in the field.

These and other objects, features, and advantages of the presentinvention will become apparent with reference to the followingdescription and drawing.

BRIEF DESCRIPTION OF THE DRAWING

The drawing consists of multiple figures, in which:

FIG. 1 is a schematic depiction of the formation of an amino derivativeof the representative nitroaromatic compound "dinoseb" under aerobicconditions and the subsequent aerobic polymerization of aminoderivatives into undesirable humic-like compounds retaining the latenttoxicity of dinoseb.

FIG. 2 is a graph showing the rate of biodegradation of dinosebaccording to the present invention as a function of temperature.

FIG. 3 is a graph showing the relationship to dinoseb biodegradationrate of various concentrations of sugar (carbon), and ammonium chloride(nitrogen) for the anaerobic consortium of microorganisms.

FIG. 4 is a graph showing the rate of biodegradation of dinosebaccording to the present invention as a function of pH.

FIG. 5 is a graph showing the kinetics of anaerobic biodegradation ofdinoseb by the anaerobic consortium to intermediary compounds and thesubsequent disappearance of the intermediary compounds.

FIG. 6 is a graph similar to FIG. 5 showing the kinetics of anaerobicbiodegradation of 4,6-dinitro-o-cresol by an anaerobic consortiumpreviously acclimated to dinoseb.

FIG. 7 is a graph similar to FIG. 5 showing the kinetics of anaerobicbiodegradation of 3,5-dinitrobenzoate by an anaerobic consortiumpreviously acclimated to dinoseb.

FIG. 8 is a graph similar to FIG. 5 showing the kinetics of anaerobicbiodegradation of 2,4-dinitrotoluene by an anaerobic consortiumpreviously acclimated to dinoseb.

FIG. 9 is a graph similar to FIG. 5 showing the kinetics of anaerobicbiodegradation of 2,6-dinitrotoluene by an anaerobic consortiumpreviously acclimated to dinoseb.

FIG. 10 is a graph similar to FIG. 5 showing the kinetics of anaerobicbiodegradation of dinitrophenol by an anaerobic consortium previouslyacclimated to dinoseb.

FIG. 11 is a schematic depiction of a reaction pathway for the anaerobicbiodegradation of dinoseb according to the process of the presentinvention.

FIG. 12 is a graph showing the effect of BESA, an inhibitor ofmethanogenesis, on the anaerobic biodegradation of dinoseb by ananaerobic consortium.

FIG. 13 is another schematic depiction of a reaction pathway for theanaerobic biodegradation of dinoseb according to the process of thepresent invention.

FIG. 14 is a representative schematic depiction of a process accordingto the present invention as it would be conducted at a field site.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT I. Selection andIsolation of Dinoseb-Degrading Microorganisms

Various microorganisms capable of at least transforming dinoseb werepreliminarily selected and enriched using a chemostat having a 1-Lcapacity vessel and agitation capability (Series 500 Fermenter, L. H.Fermentation, Hayward, Calif.). An approximately 250 mL volume of 3 mmdiameter glass beads were placed in the bottom of the chemostat vesselto act as a soil-holding matrix. As a result, both aerated (supernatantliquid) and non-aerated (soil sediment) enrichment conditions weresimultaneously maintained in the chemostat.

As used herein, "transformation" of dinoseb or other nitroaromaticcompound means a chemical change other than degradation. The simplestway to confirm transformation of dinoseb is to observe the disappearanceof the bright yellow color of the compound. Under aerobic conditions,dinoseb is transformed into an amino form that subsequently becomespolymerized by oxidative coupling. The term "degradation" denotes thecomplete mineralization of the subject nitroaromatic to methane, carbondioxide, and acetate.

To provide a source of mineral nutrients to the microorganisms in thechemostat, a mineral nutrient solution was prepared containing thefollowing solutes: KH₂ PO₄ (272 mg/L), K₂ HPO₄ (348 mg/L), Na₂ SO₄ (5mg/L), MgSO₄.7H₂ O (5 mg/L), CaCl₂.2H₂ O (1 mg/L), and FeSO₄ (0.5 mg/L).The nutrient solution was supplemented with selected carbon and nitrogensources, as discussed further below, in an attempt to effect asatisfactory preliminary selection of dinoseb-degrading microorganisms.The initial inoculum of microorganisms consisted of indigenous microbesassociated with a 200-gram sample of a soil mixture removed from a sitepreviously exposed to dinoseb and suspected of having dinoseb-degradingactivity. The chemostat was operated at a flow rate of 10 mL/hr, pH 7,and 25° C. Carbon and nitrogen sources for the microorganisms usedindividually in separate attempts at selection included: 50 ppm dinosebplus 50 ppm 2,4-dinitrophenol, 50 ppm dinoseb plus 50 ppm2,4-dinitrophenol plus 1 g/L NH₄ Cl, 50 ppm dinoseb plus 50 ppm phenol,and 100 ppm dinoseb plus 0.5 g/L glucose and 1 g/L NH₄ Cl.

No dinoseb degradation or turbidity occurred in the chemostat duringthirty days' incubation and agitation with 50 ppm dinoseb plus 50 ppm2,4-dinitrophenol as the sole carbon substrate. When the chemostat wasoperated for another thirty days with 50 ppm dinoseb plus 50 ppm2,4-dinitrophenol and 1 g/L NH₄ Cl, no dinoseb degradation or turbidityresulted. When the chemostat was operated for forty-two days with 50 ppmdinoseb, 50 ppm 2,4-dinitrophenol; and 50 ppm phenol, no dinosebdegradation occurred, but turbidity did develop. Most importantly, whenthe chemostat was operated with 0.5 g/L glucose, 100 ppm dinoseb, and 1g/L NH₄ Cl, turbidity developed immediately and dinoseb degradationbegan after twenty days. The latter result indicated that, to effectdegradation of dinoseb by either aerobic or anaerobic processes,supplementary glucose is required as a carbon source. Continuing withthe latter operational conditions, the flow rate was then increased to20 mL/hr for another thirty days to complete the preliminary selectionprocess.

After preliminary selection, samples of the supernatant liquid presumedto contain various aerobic and/or facultative microorganisms wereremoved from the chemostat and plated on nutrient agar to obtain anumber of isolates of specific microorganisms. The isolates were thencultured under aerobic, anaerobic, and microaerophilic conditionswherein their individual abilities to transform or degrade dinoseb wereevaluated. For aerobic culturing, the mineral nutrient solutiondescribed above was used, supplemented with the following: dinoseb (50mg/L), glucose or fructose (500 mg/L), N₄ Cl (1.0 g/L), MnCl₂.4H₂ O (0.5mg/L), H₃ BO₃ (0.05 mg/L), ZnCl₂ (0.05 mg/L), CuCl₂ (0.03 mg/L), Na₂MoO₄.2H₂ O (0.01 mg/L), CoCl₂.6H₂ O (0.5 mg/L), NiCl₂.6H₂ O (0.05 mg/L),Na₂ SeO₃ (0.05 mg/L), and a vitamin solution recommended by Wolin etal., J. Biol. Chem. 238:2882-2886 (1963). Yeast extract was added to theculture solution to a concentration of 0.5 g/L. Yeast extract served asa convenient source of additional carbon and energy for themicroorganisms, as well as a source of additional vitamins andcofactors.

For culturing under denitrifying (anaerobic) conditions, the aerobicculture medium was supplemented with 1 g/L KNO₃, boiled under nitrogengas, and sealed in glass containers with butyl rubber stoppers beforeinoculation. Culturing under denitrifying conditions is a way ofselecting for facultative anaerobes. Under such conditions, nitrate isemployed by the respiring microbes as an electron acceptor rather thanoxygen as used by respiring aerobic microorganisms. Facultativeanaerobes can be cultured in either aerobic or anaerobic environments.

A reduced anaerobic mineral medium (RAMM) was used for culturing themicroorganisms under anaerobic conditions. RAMM comprised the sameingredients as listed above for aerobic cultures, but with 10 mg/Lresazurin added as a redox indicator, 10 mg/L NaMo₂ O₄.2H₂ O added as areducing agent, and 1.2 g/L NaHCO₃. (Preferably, 0.1 g/L yeast extractis also added.) Anaerobic cultures were grown in serum bottles and balchtubes sealed with butyl rubber stoppers, using strict anaerobicprocedures, as detailed in Ljungdahl and Wiegel, "Working with AnaerobicBacteria," in Demain and Solomon (eds.), Manual of IndustrialMicrobiology and Biotechnology, American Society for Microbiology,Washington, D.C. (1986) pp. 84-96.

Several isolates of microorganisms which could aerobically transformdinoseb were obtained from the supernatant liquid of the chemostatcontaining glucose, dinoseb, and NH₄ Cl. Characteristics of theseisolates are presented in Table 1.

                                      TABLE 1                                     __________________________________________________________________________    Isolates That Aerobically Transform Dinoseb                                       Gram     Colony.sup.a   Dinoseb Transformation When:                      Isolate                                                                           Reaction                                                                           Shape                                                                             Morphol.                                                                           Oxidase                                                                            Catalase                                                                           Facultative                                                                         microaerophilic                                                                       denitrifying                        __________________________________________________________________________    TDN-1                                                                             +    rods                                                                              R, O, P                                                                            +    +    no    yes     no                                  TDN-2                                                                             -    rods                                                                              I, T +/-  +    yes   yes     yes                                 TDN-3                                                                             -    rods                                                                              R, T -    +    yes   yes.sup.b                                                                             no.sup.b                            TDN-4                                                                             +/-  rods                                                                              R, Y +    +    yes   yes     no                                  TDN-5                                                                             +/-  cocci                                                                             S, T +/-  +    yes   yes     yes                                 __________________________________________________________________________     .sup.a Colonial Morphologies: R = round, I = irregular, S = spreading, T      transparent, O = opaque, Y = yellow, P = produces yellowgreen pigment.        .sup.b Dinoseb transformation in this strain is inhibited by nitrate     

Under microaerophilic conditions, every strain caused thedinoseb-containing culture medium to turn bright red. Suchtransformation was not noted with any of the strains in aerateddinoseb-containing cultures. As used herein, the term "microaerophilicconditions" refers to a culture environment having an appreciablydepressed concentration of dissolved oxygen as compared to aerobicconditions, but not so low as to be strictly anaerobic. Under suchconditions, aminoaromatics can still undergo polymerization reactions.This is in contrast to strictly anaerobic conditions under which suchpolymerization reactions are blocked. Microorganisms termed"microaerophiles" undergo optimal growth under microaerophilicconditions.

The red substance obtained under microaerophilic conditions with all thestrains could not be extracted with organic solvents, nor could it beresolved by thin-layer chromatography (TLC). Analysis suggested that thered substance contained the amino derivatives of dinoseb. After two tothree weeks, the red color faded and a brown precipitate formed. TLCanalysis of the brown precipitate showed a continuous smear with nodiscernable bands. These results indicate that dinoseb is transformedunder microaerophilic conditions to an amino form which is oxidativelypolymerized to larger random-length molecules and not degraded.

Strains TDN-2 and TDN-5 appeared to be facultative anaerobes and wereable to carry out the transformation of dinoseb to the red metaboliteunder denitrifying (anaerobic) conditions, in which case the brownprecipitate did not form.

To quantify the transformation of dinoseb by the strains of Table 1,dinoseb concentrations were determined by High Performance LiquidChromatography (HPLC) using a binary gradient of 10% tetrahydrofuran andmethanol (solution A) and 1% acetic acid and water (solution B) on a250×2 mm Phenomenex "Spherex" 5 μm C18 reverse-phase column (PhenomenexCorp., Rancho Palos Verdes, Calif.). A Hewlett-Packard Model 1090Ainstrument, equipped with a diode-array detector and a computerized datasystem, was used for the analyses. The solvent flow rate was 0.4 mL/min,and the column temperature was 40° C. The gradient program was aten-minute gradient from 60% solution A plus 40% solution B to 100%solution A, followed by five minutes at 100% solution A. Detection ofdinoseb and transformation products thereof was by use of thediode-array detector, measuring UV absorption at 268, 225, and 385 nm,with continuous scanning of the absorption spectrum from 190 to 450 nm.

HPLC analysis of the culture medium from strains TDN-2 and TDN-5, ofTable 1 also showed accumulation of a single dinoseb-transformationproduct with a HPLC retention time of 2.05 minutes. Dinosebtransformation by strain TDN-3 was inhibited by nitrate in bothmicroaerophilic and denitrifying conditions. No dinoseb transformationoccurred in anaerobic cultures of TDN-4, but transformation did occurwhen the flasks were opened and the cultures exposed to air. StrainTDN-1 was obligately aerobic and only transformed dinoseb undermicroaerophilic conditions.

As indicated in Table 1, these isolates of dinoseb-transformingmicroorganisms were taxonomically diverse, although no definite speciesidentifications were made. Despite this apparent species diversity, theisolates appeared to carry out similar reactions when transformingdinoseb.

The above results indicate that, in oxygen-containing environments, theisolates obtained from the aerobic supernatant of the chemostat onlyreduced the nitro groups of dinoseb, thereby forming amino products thatwere subsequently oxidized by extracellular enzymes to form amorphouspolymeric compounds, as diagrammed in FIG. 1. Since the dinoseb isapparently not actually degraded via such an aerobic process, but merely"transformed" into an amorphous polymer, the products of the processprobably latently retain all the toxicity of dinoseb. Thus, it appearsthat aerobic bacteria such as certain of the strains listed in Table 1do not actually detoxify dinoseb and would, therefore, not beappropriate for use in bioremediation of nitroaromatic contaminants inwastewaters or contaminated soils.

Although the above results were useful in elucidating the mechanism ofdinoseb transformation in aerobic environments, it became clear-that abiological method for degrading dinoseb and related nitroaromaticcompounds from contaminated soils and waters must include the use ofmicroorganisms selected in a dinoseb-containing anaerobic environment.As a result, microorganisms resident in the anaerobic sediment of thechemostat supplied with medium containing 100 ppm dinoseb plus 0.5 g/Lglucose and 1 g/L NH₄ Cl were cultured and tested.

A consortium (stable mixed population) of anaerobic microorganismscapable of degrading dinoseb to non-aromatic products was enriched fromthe population of such organisms in the chemostat as follows. Sedimentfrom the chemostat consisting of soil was used to inoculate strictlyanaerobic medium comprised of the mineral nutrient solution describedabove with added 1 g/L fructose, 1 g/L NH₄ Cl, and 100 ppm dinoseb.After five weeks' incubation, the bright yellow color of the medium (dueto the presence of dinoseb) changed to a bright orange, then faded tocolorlessness followed by development of turbidity. This activity couldbe maintained in mineral medium for three or four sediment-freetransfers, but not in medium containing 0.2 g/L yeast extract or 5%rumen fluid. Sediment-free anaerobic dinoseb-degrading cultures could bemaintained indefinitely by making three transfers in mineral medium,followed by one transfer in yeast extract-containing medium, followed byfurther mineral medium transfers. After eighteen months of suchtransfers, the dinoseb-degrading cultures remained stable in the yeastextract-containing medium, which resulted in 5- to 10-fold fasterdegradation of dinoseb than in mineral medium without yeast extract.

Degradation of dinoseb to non-aromatic products by the anaerobicconsortium did not occur unless strict anaerobic procedures werefollowed during preparation of the media and during culture transfers.This indicates that actual dinoseb degradation to non-aromatic products,as opposed to mere transformation, is an anaerobic process.

Several parameters should be controlled for optimal dinoseb degradationby the anaerobic consortium. These include temperature, nitrogen and pH.

As shown in FIG. 2, the optimal temperature for dinoseb degradation isabout 25° C. Although FIG. 2 only covers a range from 20° C. to 40° C.,a useful temperature range would be within the range for mesophilicmicroorganisms, generally between 10° C. and 40° C. Temperatures higherthan about 40° C. would either kill important microorganisms or shutdown key enzymatic reactions. Lower temperatures within this range,including temperatures within the range 10° C. to 20° C., would merelyresult in a slower metabolic rate of the microorganisms, the rategenerally dropping by about half for every ten-degree drop intemperature.

As shown in FIG. 3, the optimal sugar concentration (either fructose,glucose, or other simple sugar is suitable) is about 0.5 g/L, asindicated by the "carbon" line. The optimal NH₄ Cl concentration isabout 4 g/L, as indicted by the "nitrogen" line. However, the anaerobicculture is largely insensitive to NH₄ Cl concentration between about 1and 5 g/L. NH₄ Cl serves as an important source of nitrogen for theanaerobic microorganisms. Other nitrogen-containing compounds can alsobe used, so long as the nitrogen is in a form such as ammonium ion oramino groups. The nitrogen source should not be a nitrate becausenitrates may inhibit the process of nitroaromatic degradation by thesemicroorganisms.

Finally, the optimal pH is about 7, as shown in FIG. 4, but theanaerobic culture appears to be largely insensitive to pH values between6 and 8.

The anaerobic consortium degraded dinoseb via a series of intermediatearomatic products (A, B, C, and D, as shown in FIG. 5) which could bedetected by HPLC. As determined by UV absorption spectra, no furtheraromatic products could be detected after thirty days, indicating thatcomplete cleavage of the aromatic ring occurred. Such aromatic cleavageis a key step in the degradation of dinoseb to non-toxic compounds.

The stable consortium of anaerobic microorganisms contained at leastthree bacterial morphologies, including: short coccobacilli, 1-1.5 μmlong; medium-sized rods, 0.75×2 μm; and large rods, 1-1.5×4 μm. Brieflyexposing the consortium to air before anaerobic incubation resulted inelimination of the large rods. Also, dinoseb degradation did not proceedbeyond intermediate D (FIG. 5). When the anaerobic consortium was usedto inoculate dinoseb-containing aerobic media, a single bacterialspecies grew. That species was a gram-negative rod which grew as acoccobacillus under anaerobic conditions. The coccobacillus transformeddinoseb to a single uncharacterized product in aerobic cultures and to adifferent product in anaerobic cultures. The bacterium also was catalasepositive, oxidase negative, and most closely matched Klebsiella oxytoca(similarity index 0.677), using the BioLog GN identification system(Hayward, Calif.). The BioLog GN identification system is a method oftesting bacteria for utilization of ninety-five different carbonsubstrates, where the resulting pattern growth is compared with adatabase of patterns for known species of bacteria.

The anaerobic consortium was tested for its ability to degrade othernitroaromatic compounds. Utilization of other nitroaromatic substrateswas determined by growing cultures in medium similar to that describedabove, but with 50 ppm of the appropriate nitroaromatic compoundsubstituted for dinoseb. The anaerobic consortium was able to completelydegrade 4,6-dinitro-o-cresol (DNOC, FIG. 6) and 3,5-dinitrobenzoate(3,5-DNB, FIG. 7) to non-aromatic compounds. 2,4-Dinitrotoluene(2,4-DNT, FIG. 8) and 2,6-dinitrotoluene (2,6-DNT, FIG. 9) were degradedto intermediate products, but it was unclear whether or not the productswere eventually degraded to non-aromatics. After sixty days, theconcentration of 2,4-dinitrophenol (2,4-DNP, FIG. 10) began to decline,but the parent compound persisted for at least four months in thesecultures. Degradation of these other nitroaromatic compounds was muchslower than for dinoseb.

A dinoseb anaerobic degradation pathway consistent with the aboveresults is shown in FIG. 11.

When bromoethanesulfonic acid (BESA), a specific inhibitor ofmethanogenesis, was added to the anaerobic consortium at 200 μMconcentration, dinoseb degradation was slowed and products C and Daccumulated (FIG. 12). These products remained in the culture medium forat least another three months. Accumulation of C indicated that D isformed from C and that BESA at least partially blocked formation of D.Therefore, the reaction from C to D is probably a hydrogen-generatingreaction. Since the reaction results in an increase in hydrophobicity,it probably involves removal of one or both acetamide groups.

The anaerobic dinoseb-degradation intermediates were tentativelyidentified as follows. Anaerobic dinoseb cultures were extracted andsubjected to TLC, as described above, during both the early stages(orange color) and later stages (colorless) of dinoseb degradation.Extracts from the early stages yielded two TLC bands which were notpresent in uninoculated controls. GCMS analysis indicated that bothbands contained multiple compounds. Similar analysis of the extract fromthe later stages of degradation yielded one band not present inuninoculated controls. The mass spectra showed fragmentation patternssimilar to that for dinoseb, confirming that they correspond to dinosebderivatives. The intermediates as identified and/or hypothesized areshown in FIG. 11.

The molecular formulas of the intermediates, based on isotope abundancecalculations from the molecular weights (±0.005 AMU), are shown in Table2. The intermediate with molecular weight 220 could not be assigned amolecular formula; however, the stated mass was expected from2-sec-butyl-4-nitro-6-aminophenol. The major product in the later stagesof degradation was identified as 2-aminobenzoic acid (anthranilic acid)by GCMS comparison with authentic standards.

                  TABLE 2                                                         ______________________________________                                        Accurate Mass Determinations and Molecular                                    Formulas for Compounds Detected by GC/MS                                             molecular standard molecular                                                                              deviation from                             source mass      deviation                                                                              formula  observed mass                              ______________________________________                                        dinoseb                                                                              240.0699  .0084    C.sub.10 H.sub.12 N.sub.2 O.sub.5                                                      .0047                                      band 1 210.0891  .0063    NR                                                  band 1 220.0998  .0081    NR                                                  band 1 264.0995  .0065    NR                                                  band 2 234.0966  .0071    C.sub.12 H.sub.14 N.sub.2 O.sub.3                                                      .0038                                      band 2 165.1147  .0046    C.sub.10 H.sub.15 NO                                                                   .0007                                      band 1B                                                                              136.060   .0088    C.sub.7 H.sub.8 N.sub.2 O                                                              .0035                                      band 1B                                                                              137.0508  .0047    C.sub.7 H.sub.7 NO.sub.2                            ______________________________________                                         NR = no reasonable formula could be calculated from this mass reading    

Under anaerobic conditions the reduced dinoseb derivatives would not besubject to the oxidative coupling reactions found in the aerobicenvironment. Partially degraded molecules persisting as dissolvedmonomers are, therefore, available for further biodegradation by variousanaerobic bacteria.

It is unclear why an external carbon source is required for dinosebmineralization by the anaerobic consortium of microorganisms. One ormore of these microorganisms may effect certain chemical changes to thedinoseb molecule, but derive no metabolic energy from such reactions orfrom the products of such reactions. Alternatively, microorganisms thatcarry out later steps in the degradation process might be inhibited bydinoseb. In the latter case, ring cleavage products would not beavailable for use by bacteria carrying out early steps in thedegradation process.

The results described above indicate, therefore, that dinoseb isbiodegradable by the anaerobic consortium of microorganisms. The initialstep involving reduction of nitro groups to amino groups appears tooccur in a manner similar to that for rumen microorganisms.Intermediates detected having molecular weights of 220, 234, and 264were not identified, but probably represent N-alkylated forms of reduceddinoseb. The alkyl side chain of dinoseb is also anaerobically removed,as indicated by formation of anthranilic acid. FIG. 13 shows a dinosebdegradation pathway consistent with the results above.

Studies in which [¹⁴ C]-ring-labeled dinoseb was degraded as aboveyielded [¹⁴ C]-acetate, indicating that dinoseb rings are cleaved.Additionally, we have found that the presence of nitroaromatics inhibitsmethanogenesis. However, the nitro groups are readily reduced to aminogroups, after which methanogenesis resumes. The only intermediatesidentified to date are amino compounds, but numerous other"intermediates" have been found that remain to be specificallyidentified.

When tested with other nitroaromatic substrates, the anaerobicconsortium was able to completely degrade nitrocresols ornitrobenzoates, but was unable to cleave the aromatic ring ofnitrotoluenes or to degrade nitrophenols. It is probable that otherselections performed in a manner similar to that described above wouldgive rise to other anaerobic populations which can utilize thesecompounds. For example, passing a medium containing a nitrophenoliccompound through a chemostat, as described above, inoculated with adiverse population of soil microorganisms would be expected to selectfor anaerobes capable of mineralizing nitrophenols. In other words,anaerobic consortia can be "tailored" for degrading a particular type ofnitroaromatic compound.

II. Bioremediation of Fluid Media

As used herein, the term "fluid medium" refers to waters and slurries,including mud, comprising water plus soil or other particulate material.

The process of the present invention requires an aqueous fluid mediumbecause anaerobiosis can only occur in aqueous environments. As aresult, bioremediation of a contaminated dry soil requires that water beadded, forming a mud slurry, to provide a sufficiently aqueousenvironment for metabolic activity by the anaerobic consortium ofmicroorganisms. Such an aqueous environment is automatically providedwhen the process is to be applied to a contaminated wastewater.

In addition, the anaerobic consortium is unable to withstand prolongedexposure to oxygen. Consequently, an inoculum comprised of suchmicroorganisms should not be added to a volume of soil slurry orwastewater to be subjected to biodegradation without first rendering thevolume anaerobic. The transition to anaerobiosis should be as rapid aspossible to preclude aerobic oxidative coupling of the contaminantnitroaromatics to polymeric forms via the action of indigenous aerobicmicroorganisms.

For the experiments described below, the anaerobic dinoseb-degradingconsortium isolated as described above was maintained on a reducedanaerobic mineral medium (RAMM) consisting of: KH₂ PO₄ (0.27 g/L), K₂HPO₄ (0.35 g/L), NH₄ Cl (1.5 g/L), glucose (0.5 g/L), yeast extract (0.1g/L), CaCl₂.2H₂ O (15 mg/L), MgCl₂.6H₂ O (20 mg/L), FeCl₂.2H₂ O (4mg/L), MnCl₂.4H₂ O (0.5 mg/L), H₃ BO₃ (0.05 mg/L), ZnCl₂ (0.05 mg/L),CaCl₂.2H₂ O (0.05 mg/L), NiCl₂.6H₂ O (0.05 mg/L), CuCl₂ (0.03 mg/L),NaMoO₄.2H₂ O (0.01 mg/L), NaHCO₃ (2.4 g/L), and 1 ng/L resazurin.Cultures were incubated in darkness without shaking at 30° C. Strictanaerobic procedures were followed during all media preparations andtransfer operations. Ljungdahl and Wiegel, "Working with AnaerobicBacteria," in Demain and Solomon (eds.), Manual of IndustrialMicrobiology and Biotechnology, American Society for Microbiology,Washington, D.C. (1986).

In order to sustain anaerobiosis in the present process, a source ofreadily-metabolizable carbon, such as sugar, is required as asupplement. Preferably, the carbon source is a complex carbohydrate fromwhich sugars are "released" over a period of time, rather than a supplyof sugar that is completely "available" for immediate consumption.Because complex carbohydrates must be enzymatically cleaved to yieldmetabolizable sugar, they allow maintenance of strict anaerobicconditions in the fluid medium to be extended for a time periodsufficient to biodegrade the particular concentration of contaminantnitroaromatic without the need to add more carbon source. For example,if a large amount of an "available" sugar, such as glucose or fructose,were added to an aerobic aqueous slurry of soil, anaerobiosis would bequickly achieved. However, maintenance of strict anaerobiosis for theseveral weeks that may be required to achieve complete degradation ofthe nitroaromatic would be difficult due to an initially very high rateof sugar metabolism followed by premature exhaustion of the sugarsupply.

Although sugar could be added at one or more additional times duringmaintenance of anaerobic conditions before complete nitroaromaticdegradation was achieved, the added sugar would have to be added incontrolled amounts at specific times and mixed each time into the fluidbeing treated. Such adding and mixing impart unnecessary complexity tothe method. Further, use of more complex carbohydrates, such as starch,as a carbon source results in a steadier degradation rate. Repeatedadditions of sugar result in an undulating rate which is less efficient.

In view of the above, desired characteristics of the carbon sourceinclude a high energy content, ready metabolizability, low numbers ofindigenous heterotrophic bacteria which might compete with the anaerobicinoculum, and sustained metabolic availability sufficient to maintainprolonged steady rate of anaerobiosis. On the basis of these criteria, ahydrolyzable polysaccharide such as starch is a more suitable carbonsource than free sugar.

After evaluation of a number of starchy by-products from various foodprocessing plants, a preparation of dewatered solids from a potatoprocessing plant was selected as the preferred carbon source. Thecharacteristics of the potato waste included: 42% solids, 215 mg/gavailable starch, 6.7 mg/g total nitrogen, 2.6×10⁴ culturableheterotrophic bacteria per gram, and 8×10³ culturable amylolytic (ableto hydrolyze starch) bacteria per gram. Chief advantages of starchypotato waste are available in large amounts and at a low cost. However,any vegetable or grain starch would probably suffice.

Candidate starchy carbohydrates were analyzed for solid content byweighing after oven drying, and for total nitrogen content by theKjeldahl procedure commonly known in the art. Available starch wasdetermined by incubating sterilized 1-gram samples with 300 units ofα-amylase and 100 μL of diazyme L-100 (Miles Pharmaceuticals, Elkhart,Ind.) for twenty-four hours in 10 mL of sterile 0.4M phosphate buffer,pH 7.0. After incubation, the samples were diluted to 100 mL andanalyzed for reducing sugars using the dinitrosalycilate assay withsimilarly prepared glucose as a standard. Miller, Anal. Chem. 31:426-428(1959). Available starch was assayed as mg sugar released per gram dryweight of starchy carbohydrate. Numbers and types of bacteria in starchycarbohydrates were determined by standard plate counts in aerobicmineral medium agar containing (for total heterotrophic counts) 2 gglucose, 0.4 g yeast extract, 1 g NH₄ Cl, and 1 g NaNO₃ per liter, or(for amylolytic bacteria) 2 g soluble starch, 1 g NH₄ Cl, and 1 g NaNO₃per liter for amylolytic bacteria.

Since the anaerobic consortium cannot withstand prolonged exposure toair, it is necessary to pretreat the volume of wastewater or soil slurrywith a rapid aerobic fermentation step to deplete oxygen in the volumeand achieve anaerobiosis before adding the anaerobic consortium. Thetransition from aerobic fermentation to anaerobiosis should be as rapidas possible to preclude oxidative coupling of aminoaromatic derivativesof the contaminant nitroaromatic in the aerobic step. According to theresults of tests performed using loamy sand and rich silt-loam soils,simply flooding the soil with water does not produce sufficiently rapidanaerobiosis. Mere addition of sugar produces rapid anaerobiosis by aninitial high rate of aerobic fermentation that rapidly consumes theavailable oxygen in the liquid. However, as discussed above, use ofsugar as a carbon source instead of a complex carbohydrate, such asstarch, usually results in premature exhaustion of the carbon source.Also, even with elevated concentrations of added sugar, at leastpartially aerobic conditions often reform before completion of thenitroaromatic degradation. Use of dewatered potato solids or otherstarchy carbohydrate that is metabolized more slowly than free sugarresults in a satisfactorily rapid anaerobiosis following aerobicfermentation, while yielding a sustained sugar concentration over theperiod of time required to completely degrade the contaminantnitroaromatic.

To evaluate the relative effectiveness of starchy carbohydrates as acarbon and energy source versus a sugar such as glucose alone, 300-gsamples of various types of soil, such as loamy sand and rich silt-loam,were individually placed in 1-liter Erlenmeyer flasks and flooded with200 mL of 0.4M phosphate buffer (pH 7) to form a mud slurry. Flasks werecovered with aluminum foil and incubated without shaking at 25° C. Atvarious times during incubation, both pH and redox potential of theslurry were measured potentiometrically at 0, 1, and 3 cm beneath theliquid surface. A 1 mL sample was also removed for each analysis ofresidual sugar. A redox potential of -200 mV or less was indicative of astrictly anaerobic condition in the liquid.

The amount of starchy carbohydrate to add to a volume of wastewater ormud slurry for particulate dosages of fermentative and anaerobicorganisms may have to be determined experimentally and optimized for aparticular soil or wastewater and the particular type and concentrationof contaminant nitroaromatic. To reduce costs in the actualbioremediation process, the amount of starch should be determined thatwill just sustain anaerobic conditions for the time required to achievethe desired degree of nitroaromatic degradation and no longer.

Any of a number of species of fermentative amylolytic microorganismsindigenously present in the starchy carbohydrate can serve tohydrolytically cleave the starch into constituent sugars when the starchis added to a volume of wastewater or slurry of soil. Such fermentativemicroorganisms would include aerobic and/or facultative microorganisms.Starch cleavage occurs via amylase enzymes secreted by themicroorganisms into the surrounding aqueous medium. Although amylaseaction is optimal in aerobic environments, the enzyme will continue tohydrolyze starch when the medium becomes anaerobic. Additionally, manyfacultative amylolytic microorganisms will survive and continue tosecrete amylase after a medium has been converted from aerobic toanaerobic. If sugar is used as the carbon source, the fermentativemicroorganisms need not be amylolytic.

In a series of incubation tests where the soil contained 25 ppm dinoseb,anaerobiosis occurred only after several days in the presence of thestarchy potato by-product, where the initial aerobic fermentation wasperformed by fermentative amylolytic bacteria (representing a number ofspecies) indigenous to the starchy potato by-product. Several days toachieve anaerobiosis is too long. Evidently, the indigenous bacterialpopulation was adversely stressed by the dinoseb and unable tometabolically respond in a rapid manner. To selectively enrich fordinoseb-resistant amylolytic bacteria, additional starchy potatoby-product and dinoseb (to 100 ppm) were added to a flask from theseexperiments. After a prolonged incubation to achieve selection, severaldinoseb-resistant bacteria were isolated from the flask. One strain(strain DSA-1), identified as facultative Klebsiella oxytoca by theBioLog GN system, retained good amylolytic activity in the presence of100 ppm dinoseb.

When dinoseb-containing soil received an inoculum of Klebsiella oxytocastrain DSA-1, along with the starchy potato by-product, a greatlyreduced time to achieve anaerobiosis was noted. In the latter case,anaerobiosis was established almost as fast as in control soils lackingdinoseb. Klebsiella oxytoca strain DSA-1 was thus regarded as apreferred inoculation strain with which to achieve anaerobiosis fordegrading dinoseb in the presence of a starchy carbohydrate.

Although Klebsiella oxytoca strain DSA-1 was the particular bacteriumselected for in the above-described experiments involving dinoseb, it isexpected that other selections performed in a similar manner using asource of starch containing indigenous aerobic and/or facultative"fermentative" microorganisms will probably yield other species andstrains after selection. Additionally, it is expected that using anitroaromatic other than dinoseb for supplying the selection pressurewould probably give rise to other satisfactory fermentative speciesand/or strains resistant to the particular nitroaromatic. The lessonfrom these studies is that the indigenous microflora associated withstarches are probably not sufficiently tolerant to most nitroaromaticsto facilitate the required high initial rate of fermentation to achieverapid anaerobiosis in a volume of soil slurry or wastewater containingnitroaromatic compounds. As a result, it will probably be necessary toemploy an aerobic or facultative species and strain preselected againstthe particular nitroaromatics to be biodegraded.

In order to achieve anaerobiosis in the shortest amount of time, it ispreferable to inoculate using a large dose of preselected fermentativemicroorganisms. Our studies indicate that a dose of about 10⁷ to 10⁸ CFUper gram dry soil or mL water is an effective dose. Cost constraintsgenerally preclude larger doses. Also, larger doses generally do notachieve proportionately shorter times to anaerobiosis.

In another series of tests, bioremediations of -kg samples of each typeof dinoseb-contaminated soil (loamy sand and silt-loam) wereindividually performed by adding water, starchy potato by-product, andpretreating the resulting slurry with a large inoculum (as above) ofKlebsiella oxytoca strain DSA-1. After each slurry was renderedanaerobic (redox potential (-200 mV), it was inoculated with a similarlylarge dose of the dinoseb-degrading consortium of anaerobicmicroorganisms. Dinoseb was found to be completely converted tointermediate compounds within one week of anaerobic inoculation. Afterfour weeks, the concentrations of the intermediate compounds declinedbelow detectable limits. At the end of the four-week period, only 0.5ppm of dinoseb could be Sohxlet-extracted from the treated loamy sandsoil, and none could be extracted from the treated silt loam. Bycontrast, in the uninoculated loamy sand control, dinoseb did notdecline. In the uninoculated silt loam control, dinoseb did declineafter several days' lag time, but no intermediate compoundsmetabolically derived from dinoseb were detected.

In similar experiments, soils contaminated with like concentrations of4,6-dinitro-o-cresol and 3,5-dinitrobenzoate were bioremediated withinthirty days.

To perform the above experiments, soils were adjusted to 100 ppm dinosebbefore adding any microorganisms or starchy carbohydrate by adding tothe soil a solution of dinoseb in methanol and allowing the soil to drycompletely afterward. For each soil sample, 1 kg dinoseb-supplementedsoil was mixed with 2 g of the starchy carbohydrate. The mixture wasinoculated with the amylolytic bacteria, placed in an open two-litercapacity Erlenmeyer flask, and a sufficient volume 0.4M phosphate buffer(pH 7) was added to saturate the water-holding capacity of the soil.When the redox potential of the soil solution became less than -200 mV,a 50 mL volume (O.D.≈1) of the anaerobic consortium was injected belowthe surface of the soil. Samples (approximately 5 g each) were removedperiodically, weighed and vortexed with 5 mL 0.1N NaOH. Samples werecentrifuged to remove the solids, and the supernatant was analyzed fordinoseb and its metabolites. At the end of the incubation, the remainingsoil was extracted and analyzed for dinoseb.

Dinoseb concentrations were determined by high performance liquidchromatography (HPLC) using a binary gradient of 10% tetrahydrofuran inmethanol (solution A) and 1% acetic acid in water (solution B) on a250×2 mm Phenomenex "Spherex" 5 μm C18 reverse-phase column. Analyseswere performed using a Hewlett-Packard Model 1090A instrument, asdescribed above. Dinoseb was extracted from soils by Soxhlet extractionwith ethyl acetate for five hours. Before extraction, each sample wasamended with 200 μL of a 0.25% solution of 4,6-dinitro-o-cresol inmethanol, which served as an extraction standard, and lyophilized.Samples were then amended with 100 μL of 0.01M H₂ SO₄. After extraction,the extracts were dried over anhydrous Na₂ SO₄, evaporated under avacuum, and dissolved in 5 mL of ethyl acetate.

Parallel experiments utilizing [⁻ C]-dinoseb were performed to evaluatemass-balance relationships for dinoseb and its degradation products.Details are described below. Results are shown in Table 3.

                  TABLE 3                                                         ______________________________________                                        Percent of Total Radioactivity                                                                 Polar   Nonpolar     Percent                                 Treatment                                                                              CO.sub.2                                                                              Extract Extract Solid                                                                              Recovery                                ______________________________________                                        Loamy Sand                                                                             29.6    43.3    5.11    4.0* 82                                      Inoculated                                                                    Loamy Sand                                                                             0.3     0.8*    100.8   6.7* 108                                     Uninoculated                                                                  Silt Loam                                                                              32.1    29.9    3.5     8.9* 74                                      Inoculated                                                                    Silt Loam                                                                              0.8     1.1*    31.3    44.8 78                                      ______________________________________                                         *these values were not significantly different from background counts at      the 90% confidence level                                                 

Referring to Table 3, about 30% mineralization to [¹⁴ C]-CO₂ wasobtained after thirty days' incubation of samples of inoculated soils,as compared to less than 1% for the uninoculated controls. Most of theremaining radioactivity in the inoculated soils was present as polarmetabolites. Less than 10% of the radioactivity was associated withsolids in the inoculated soils after extraction, as compared to nearly45% in the silt-loam control. In the loamy sand control, virtually allof the radioactivity was associated with the nonpolar extract, whichcontained undegraded dinoseb. In addition, although methane was notquantified in these experiments, some radioactivity appears to end up asmethane.

To perform the radiochemical studies described above, radiolabeleddinoseb (u-ring [¹⁴ C]-dinoseb) was synthesized from [¹⁴ C]-phenol andwas 96% radiochemically pure, as determined by HPLC analysis and TLCcoupled with liquid scintillation counting. After adding 1 μCi u-ring[¹⁴ C]-dinoseb to the soil, the flasks were stoppered and a glass trapcontaining 1 mL of 1M KOH was suspended in each flask. The KOH solutionwas exchanged daily during incubations, rinsed with 1 mL water andcounted with 18 mL of BioSafe II liquid scintillation cocktail (RPIInc., Mt. Prospect, Ill.). At the end of the incubation, each soilculture was connected to a CO₂ trap consisting of a series of fourstoppered serum bottles each containing 10 mL of 1N KOH. The gaseouseffluent first passed through a C18 "Sep-Pak" cartridge (WatersAssociates, Milford, Mass.) wetted with methanol to remove volatileorganics. Each soil culture was then acidified with 10 mL concentratedHCl, agitated, and flushed with nitrogen gas to drive off dissolved CO₂.One-mL samples from each trap were counted with 19 mL of BioSafe II.

Three 25-gram subsamples of each soil slurry were neutralized andextracted with 0.1N NaOH. The extracts were neutralized, then passedthrough a "Sep-Pak" C18 cartridge which was rinsed with 1 mL water. Thesolid subsamples were dried, Soxhlet extracted, and the extracts werepooled with ethylacetate elutions from the "Sep-Pak" cartridges. Fromboth the polar and non-polar extracts, 1 mL samples were counted with 19mL of BioSafe II. Finally, 0.25 gm of each extracted soil sample wasmixed with 19 mL BioSafe II and counted. All radioactive samples werecounted in a Beckman Model 7000 liquid scintillation counter. A controlflask for each soil type was treated similarly except that no bacterialinoculations were made.

In the above experiments, simply flooding the soils did not produceanaerobiosis in the resulting soil slurries, even in a slurry of therich silt-loam soil. It is probable that anaerobiosis occurred withinhighly localized sites within the slurry, such as soil pores, but thiswas not sufficient to support growth of the oxygen-sensitive anaerobicconsortium. In every case, exogenous carbon, preferably as a starchycarbohydrate metabolized by amylolytic microorganisms, was required toproduce the requisite strict anaerobiosis that would allow growth of thesubsequently added dinoseb-degrading anaerobic microorganisms.

The experiments described above show that exogenous, strictly anaerobicmicroorganisms can be used to degrade nitroaromatic chemicals in soil orwastewater. The mass balance and ¹⁴ C data indicate that the anaerobicconsortium mediated a complete destruction of dinoseb as arepresentative nitroaromatic, rather than mere polymerization of thecompound, which occurred in the silt-loam soil in the absence ofanaerobic inoculation.

The method of the present invention, therefore, comprises basically twostages: an initial fermentative stage wherein the wastewater or aqueousslurry of nitroaromatic-contaminated soil is fortified with a starchycarbohydrate and inoculated with "fermentative" (aerobic and/orfacultative) amylolytic bacteria that hydrolyze the starch, metabolize aportion of the sugars produced by the hydrolysis, and consume the oxygenin the liquid; and a subsequent anaerobic stage wherein the wastewateror aqueous slurry of contaminated soil is inoculated with a consortiumof anaerobic bacteria that metabolize the remaining sugar and degradethe contaminant nitroaromatic. This bioremediation method may be usedfor any soil or water contaminated with dinoseb or other nitroaromaticor aminoaromatic compounds subject to polymerization reactions inaerobic soil, such as 4,6-dinitro-o-cresol and 3,5-dinitrobenzoate.

Although anaerobiosis can be performed in an open environment, it isinefficient and difficult to control unless performed in a closedenvironment, particularly on a large scale. As a result, the method ofthe present invention is performed in the field, preferably in asuitably large liquid-containment vessel or the like.

FIG. 14 is a schematic representation of one embodiment of the processof the present invention, as it would be conducted in the field tobioremediate a nitroaromatic-contaminated soil. In FIG. 14, thecontaminated soil 22 is transferred via equipment 24 to a plastic-linedpit 26 or the like. After the soil 22 is added to the pit 26, starchycarbohydrate 30, a nitrogen source 32, water 34, and an inoculum offermentative amylolytic microorganism 36 are added to the soil 22,forming a slurry 28 in the pit 26. After forming the slurry 28, a cover38 is placed over the pit 26, where the plastic-lined pit 26 and cover38 together serve to exclude gaseous exchange between the slurry 28 andthe atmosphere. The slurry 28 is monitored using a potentiometric probe40 coupled to a readout 42 to enable one to determine when anaerobicconditions have developed in the slurry 28 (redox potential about -200mV or less). After the slurry 28 reaches anaerobiosis, an inoculum of ananaerobic consortium 44 of microorganisms is added to the slurry 28beneath the surface, after performing sufficient numbers of serialtransfers 46 of the anaerobic microorganisms 44 to yield a suitablylarge dose. The slurry 28 is maintained in an anaerobic condition in thecovered pit until the nitroaromatics are biodegraded, at which time theslurry 28 may be returned 48 to the original site.

The process of FIG. 14 is a batch process. In a batch process applied tonitroaromatic-contaminated wastewater (not shown), the contaminatedwater is simply conducted into the plastic-lined pit 26 of FIG. 14 oranalogous vessel for bioremediation according to the present method. Inthe case of soil, it is usually necessary to add water to the soil toform a mud slurry, which is a more suitable environment for anaerobiosisthan dry soil. Such a slurry will have a proportion of water relative tosoil of about 15% to 20% (w/w) or more, depending upon the type of soiland the degree of nitroaromatic contamination, to preferably bring thesoil at least to about 100% water-holding capacity. More water can beadded, if desired. More water added to the soil lessens the effectiveconcentration of the contaminant nitroaromatic.

Since most soils and many natural waters already have a broad spectrumof mineral nutrients available to support life, in many cases it willnot be necessary to add to the water or mud slurry many of thesupplementary mineral nutrients required for laboratory cultures. Invirtually all cases, however, an extraneous nitrogen source 32 will berequired, such as ammonium chloride. Many soils and most waters willalso require extraneous phosphate (not shown), such as sodium orpotassium phosphate. The nitrogen and phosphate requirements for thepresent process can be satisfied in many cases by merely adding ammoniumphosphate (a common "fertilizer") as a supplement until the approximatedesired concentration of ammonium and phosphate are attained. Since eachsoil and water is different, preliminary analysis of the soil or watermay be needed to determine indigenous concentrations of ammonium andphosphate so that the proper amount of supplementary ammonium andphosphate can be determined so as to avoid waste.

As stated above, the nitrogen source 32 should probably not be nitratebecause nitrates may inhibit the ability of the anaerobic microorganismsto biodegrade nitroaromatics.

It may be necessary, particularly in the case of strongly acidic watersor soils contaminated with nitroaromatics, to adjust the pH of the fluidmedium to within the preferred range of about 6 to 8. An inexpensiveadditive, such as lime, is satisfactory for elevating the pH. In mostcases, however, significant pH adjustment will not be necessary.Extrapolating from the graph of FIG. 4, it would seem that pH valuesappreciably above or below (especially above) the preferred range of 6to 8 would not cause a catastrophic depression of microbial metabolismrequired for bioremediation.

The preferred target concentrations of key nutrients (includingcontributions by the soil and/or water) are as given in the list of suchingredients in the nutrient solutions used in the studies describedabove. The ammonium concentration, as stated earlier, should preferablybe between 0.4% and 0.5% (w/v), but a concentration between 0.1% and0.6% (w/v) would suffice. The overall ionic strength of the water orslurry to be treated should preferably be approximately that of thelaboratory cultures of microorganisms, but the microbes are tolerant tosurprisingly large variations of ionic strength.

The temperature should preferably be between about 10° C. and 40° C.,with the optimal temperature about 25° C. As discussed above, lowernon-freezing temperatures tend to slow microbial metabolism andcorrespondingly increase the length of time required to achievebioremediation.

While yeast extract is an important supplement for laboratory cultures,it is not necessary in virtually all field applications, since soils andwastewater often contain "vitamins," cofactors, and other products ofnatural organic processes utilizable by the microorganisms of thepresent process.

The inoculum of fermentative amylolytic microorganisms 36 should beadded at a dose of 10⁷ to 10⁸ CFU (colony forming units) per gram drysoil or per mL water. Higher doses are usually not necessary. Lowerdoses may result in a longer-than-optimal time to reach anaerobiosis. Inaddition, an amount of starchy carbohydrate 30, determined as describedabove, is also added. Preferably, the amount of starchy carbohydrateadded per unit amount of water or soil slurry is experimentally"tailored" to sustain anaerobiosis for a sufficient amount of time toachieve the desired degree of nitroaromatic degradation in the water orsoil. Since types and concentrations of nitroaromatic contaminants willdiffer among various soils and waters, and since soils and watersthemselves will differ, the optimal amount of starchy carbohydraterequired will probably differ at each of various contaminated sites.

While the specific anaerobic consortium 44 of microorganisms describedherein is suitable for degrading dinoseb and certain othernitroaromatics as described, other consortia of anaerobic microorganismsselected for and isolated in a manner as described herein, but using anitroaromatic other than dinoseb may be more suitable for othernitroaromatics.

Although employing fermentative microorganisms to render the water orslurry anaerobic is the preferred method, other methods may be employed(not shown). However, it is anticipated that other methods may beprohibitively expensive. Such other methods include purging oxygen fromthe liquid using, for example, nitrogen or argon gas. However,gas-purging is typically slower and less efficient in achievingsatisfactory anaerobic conditions than employing aerobic fermentation.Another method would require adding oxygen-scavenging agents (strongreducing agents) to the liquid. Although use of reducing agents may beefficient (and rapid), the disadvantage is that such agents representother contaminants added to the water or slurry. As a result, use ofmicrobial fermentation of sugar or starch is the preferred approach forachieving anaerobiosis.

After the contaminant nitroaromatic has been satisfactorily biodegradedin the volume of water or slurry contained in the covered pit 26 orvessel, the pit 26 or vessel may be drained and a new volume ofcontaminated water or mud slurry added for bioremediation. In asemicontinuous process, about 10% to 15% of the previous batch oftreated water or slurry may be left in the pit 26 or vessel to aid inthe inoculation of the subsequent batch. Additionally, such asemicontinuous process would preferably be controlled by variouscontinuous electronic and chemical monitoring techniques known in theart, such as of dissolved oxygen and specific ions, as well asenvironmental conditions such as pH and temperature. Concentrations ofnitroaromatic contaminants and their metabolic intermediaries can bediscontinuously monitored using HPLC and gas chromatography, forexample.

As discussed above, the carbohydrate-fermentative microorganismsresponsible for generation of anaerobic conditions and lowering of theredox potential of the fluid medium typically comprise various groups ofsuch microorganisms present as a consortium. Likewise, the anaerobicmicroorganisms responsible for actual degradation of nitroaromaticcompounds are represented as a consortium of microorganisms. The speciescomposition and species profiles of these consortia typically varydepending mainly upon the source of the microorganisms and upon theparticular nitroaromatic compound to be degraded. (I.e., the inocula arepreferably produced, as described above, by procedures in whichmicroorganisms from a source are subjected to a "selection" protocolinvolving exposure to the particular nitroaromatic to be degraded. Thisselection protocol as well as the inevitable variation inmicrobiological profiles of inocula obtained from different sources,result in the production of suitable inocula each represented by adistinctive species profile that functions as a consortium. Thus,various combinations of microorganisms can possess the completemetabolic capacity to degrade nitroaromatic compounds. Despite theinherent variation in the species profile of various inocula, certaingroups of microorganisms routinely appear in each type of consortium.These groups are listed in Table 4, using nomenclature as found inSneath et al. (eds.) Bergey's Manual of Systematic Bacteriology, Vol.1-4, Williams and Wilkins, Baltimore (1986).

TABLE 4

I. Microorganisms typically present in consortia used for generation ofanaerobic conditions:

A. Facultatively anaerobic Gram-negative rods of the familyEnterobacteriaceae, including genera such as Klebsiella andEnterobacter.

B. Gram-positive fermentative non-sporulating bacteria such as the genusLactobacillus.

C. Denitrifying bacteria such as the genera Bacillus, Clostridium, andPseudomonas.

II. Microorganisms typically present in the anaerobic consortium:

A. Microorganisms responsible for reduction of aromatic nitro groups toform reduced intermediates:

1. Facultatively anaerobic Gram-negative rods of the familyEnterobacteriaceae, including genera such as Klebsiella andEnterobacter.

2. Anaerobic Gram-negative straight, curved, and helical rods of thefamily Bacteroidaceae, including genera such as Bacteroides andFusobacterium.

3. Dissimilatory sulfate- or sulfur-reducing bacteria, including generasuch as Desulfovibrio and Desulfuromonas.

4. Anaerobic endospore-forming Gram-positive rods and cocci, includinggenera such as Clostridium, Desulfotomaculum, and Sporosarcina.

5. Gram-positive fermentative non-sporulating bacteria such as the genusLactobacillus.

6. Denitrifying bacteria, including genera such as Bacillus,Clostridium, and Pseudomonas.

B. Microorganisms responsible for cleaving the aromatic rings of reducedintermediates of the nitroaromatics:

1. Anaerobic Gram-negative straight, curved, and helical rods of thefamily Bacteroidaceae, including genera such as Bacteroides andFusobacterium.

2. Dissimilatory sulfate- or sulfur-reducing bacteria, including generasuch as Desulfovibrio and Desulfuromonas.

3. Anaerobic Gram-negative cocci, including genera such as Veillonellaand Acidaminococcus.

4. Anaerobic endospore-forming Gram-positive rods and cocci, includinggenera such as Clostridium, Desulfotomaculum, and Sporosarcina.

C. Microorganisms responsible for anaerobic fermentation and anaerobicrespiration and, ultimately, methanogenesis of the products of ringcleavage:

1. Anaerobic Gram-negative straight, curved, and helical rods of thefamily Bacteroidaceae, including genera such as Bacteroides andFusobacterium.

2. Dissimilatory sulfate- or sulfur-reducing bacteria, including generasuch as Desulfovibrio and Desulfuromonas.

3. Archaebacteria, including Group-I methanogenic archaebacteria;representative genera include Methanobacterium and Methanococcus.

4. Archaebacteria, including Group-II archaebacterial sulfate reducers;representative genus is Archaeoglobus.

5. Anaerobic endospore-forming Gram-positive rods and cocci, includinggenera such as Clostridium, Desulfotomaculum, and Sporosarcina.

6. Denitrifying bacteria, including genera such as Bacillus,Clostridium, and Pseudomonas.

Having illustrated and described the principles of our invention withreference to detailed descriptions of process steps and specificexamples, it should be apparent to those of ordinary skill in the artthat the invention may be modified in arrangement and detail withoutdeparting from such principles. We claim as our invention all suchmodifications as come within the true spirit and scope of the followingclaims.

We claim:
 1. A method for biodegrading a nitroaromatic compound presentas a contaminant in a sample, comprising the steps of:(a) providing asample comprising a nitroaromatic compound; (b) adding to the sample aninoculum comprising a nitroaromatic-degrading microorganism thatdegrades the nitroaromatic compound under anaerobic conditions, whereinthe nitroaromatic-degrading microorganism is of a genus selected fromthe group consisting of Klebsiella, Enterobacter, Bacteroides,Fusobacterium, Desulfovibrio, Desulfuromonas, Clostridium,Desulfotomaculum, Sporosarcina, Lactobacillus, Bacillus, Pseudomonas,Veillonella, Acidaminococcus, Methanobacterium, Methanococcus, andArchaeoglobus; (c) producing anaerobic conditions in the sample; and (d)maintaining the anaerobic conditions in the sample for a time that issufficient for the nitroaromatic-degrading microorganisms to degrade thenitroaromatic compound.
 2. The method of claim 1 wherein the inoculumfurther comprises a carbohydrate-fermenting microorganism that isresistant to the nitroaromatic compound and ferments a fermentablecompound under aerobic conditions, wherein the carbohydrate-fermentingmicroorganism is of a genus selected from the group consisting ofKlebsiella, Enterobacter, Lactobacillus, Bacillus, Clostridium, andPseudomonas.
 3. The method of claim 2 wherein step (c) comprises thesteps of:adding to the sample an amount of the fermentable compound; andmaintaining the sample under conditions suitable for fermentation by thecarbohydrate-fermenting microorganism of at least a portion of theamount of the fermentable compound, thereby consuming dissolved oxygenand producing anaerobic conditions in the fluid medium.
 4. The method ofclaim 3 wherein the fermentable compound is a carbohydrate.
 5. Themethod of claim 4 wherein the carbohydrate is a starch.
 6. The method ofclaim 1 wherein the inoculum is a consortium of microorganisms.
 7. Themethod of claim 6 wherein the inoculum is an enriched consortium ofmicroorganisms produced by culturing a naturally-occurring population ofmicroorganisms in a medium comprising the nitroaromatic compound.
 8. Themethod of claim 6 wherein the inoculum comprises an amount of a samplepreviously subjected to a nitroaromatic biodegradation treatmentutilizing microorganisms.
 9. The method of claim 1 further comprisingthe step of adding to the sample a member of the group consisting of anutritional source of nitrogen and a nutritional source of phosphorousfor the microorganisms.
 10. The method of claim 1 further comprising thestep of adding water to the sample.
 11. The method of claim 10 whereinthe sample is a member of the group consisting of a water and an aqueousslurry of soil or other particulate matter.
 12. The method of claim 1,wherein the sample is maintained at a pH within a range of about 6 to 8.13. The method of claim 1 wherein the sample is maintained at atemperature within a range of about 10° C. to about 40° C.
 14. Themethod of claim 1 wherein the sample, under the anaerobic conditions,has a redox potential of less than about -200 mV.
 15. The method ofclaim 1 further comprising the step of sequestering the sample in avessel.
 16. The method of claim 15 further comprising the step ofcovering the vessel so as to decrease gas exchange between the sampleand the atmosphere.
 17. A method for degrading a nitroaromatic compoundpresent as a contaminant in a sample comprising the steps of:(a)providing a sample comprising a nitroaromatic compound and anitroaromatic-degrading microorganism that degrades the nitroaromaticcompound under anaerobic conditions, wherein the nitroaromatic-degradingmicroorganism is of a genus selected from the group consisting ofKlebsiella, Enterobacter, Bacteroides, Fusobacterium, Desulfovibrio,Desulfuromonas, Clostridium, Desulfotomaculum, Sporosarcina,Lactobacillus, Bacillus, Pseudomonas, Veillonella, Acidaminococcus,Methanobacterium, Methanococcus, and Archaeoglobus; (b) producinganaerobic conditions in the sample; and (c) maintaining the anaerobicconditions in the sample for a time that is sufficient for themicroorganisms to degrade the nitroaromatic compound.
 18. The method ofclaim 17 wherein the sample further comprises a carbohydrate-fermentingmicroorganism that is resistant to the nitroaromatic compound andferments a fermentable compound under aerobic conditions, wherein thecarbohydrate-fermenting microorganism is of a genus selected from thegroup consisting of Klebsiella, Enterobacter, Lactobacillus, Bacillus,Clostridium, and Pseudomonas.
 19. The method of claim 18 wherein step(b) comprises the steps of:adding to the sample an amount of thefermentable compound; and maintaining the sample containing the addedcarbon source under conditions suitable for fermentation of at least aportion of the amount of the carbon source by the first microorganisms,thereby consuming dissolved oxygen and producing anaerobic conditions inthe sample.
 20. The method of claim 19 wherein the fermentable compoundis a carbohydrate.
 21. The method of claim 20 wherein the carbohydrateis a starch.
 22. The method of claim 17 wherein the sample comprises aconsortium of microorganisms that includes the nitroaromatic-degradingmicroorganism.
 23. The method of claim 17 further comprising the step ofadding to the sample a member of the group consisting of a nutritionalsource of nitrogen and a nutritional source of phosphorous for themicroorganisms.
 24. The method of claim 17 wherein the sample ismaintained at a pH within a range of about 6 to
 8. 25. The method ofclaim 17 wherein the sample is maintained at a temperature within arange of about 10° C. to about 40° C.
 26. The method of claim 17 whereinthe sample, under the anaerobic conditions, has a redox potential ofless than about -200 mV.
 27. The method of claim 17 further comprisingthe step of adding water to the sample.
 28. The method of claim 27wherein the sample is a member of the group consisting of water and anaqueous slurry of soil or other particulate matter.
 29. The method ofclaim 17 further comprising the step of sequestering the sample in avessel.
 30. The method of claim 29 further comprising the step ofcovering the vessel so as to decrease gas exchange between the sampleand the atmosphere.
 31. A method for biodegrading a nitroaromaticcompound present as a contaminant in a sample, comprising the stepsof:(a) adding to a sample comprising a nitroaromatic compound aninoculum comprising (i) a carbohydrate-fermenting microorganism that isresistant to the nitroaromatic compound and ferments a carbohydrateunder aerobic conditions, wherein the carbohydrate-fermentingmicroorganism of a genus selected from the group consisting ofKlebsiella, Enterobacter, Lactobacillus, Bacillus, Clostridium, andPseudomonas and (ii) a nitroaromatic-degrading microorganism thatdegrades the nitroaromatic compound under anaerobic conditions, whereinthe nitroaromatic-degrading microorganism is of a genus selected fromthe group consisting of Klebsiella, Enterobacter, Bacteroides,Fusobacterium, Desulfovibrio, Desulfuromonas, Clostridium,Desulfotomaculum, Sporosarcina, Lactobacillus, Bacillus, Pseudomonas,Veillonella, Acidaminococcus, Methanobacterium, Methanococcus, andArchaeoglobus; (b) adding an amount of the carbohydrate to the sample;(c) maintaining the sample under conditions suitable for fermentation ofat least a portion of the amount of the carbon source by thecarbohydrate-fermenting microorganism, thereby producing anaerobicconditions in the sample; and (d) maintaining the anaerobic conditionsin the sample for a time that is sufficient for thenitroaromatic-degrading microorganism to degrade the nitroaromaticcompound.
 32. A method for degrading a nitroaromatic compound present asa contaminant in a sample comprising the steps of:(a) providing a samplecomprising a nitroaromatic compound and (i) a carbohydrate-fermentingmicroorganism that is resistant to the nitroaromatic compound andferments a carbohydrate under aerobic conditions, wherein thecarbohydrate-fermenting microorganism of a genus selected from the groupconsisting of Klebsiella, Enterobacter, Lactobacillus, Bacillus,Clostridium, and Pseudomonas, and (ii) a nitroaromatic-degradingmicroorganism that degrades the nitroaromatic compound under anaerobicconditions, wherein the nitroaromatic-degrading microorganism is of agenus selected from the group consisting of Klebsiella, Enterobacter,Bacteroides, Fusobacterium, Desulfovibrio, Desulfuromonas, Clostridium,Desulfotomaculum, Sporosarcina, Lactobacillus, Bacillus, Pseudomonas,Veillonella, Acidaminococcus, Methanobacterium, Methanococcus, andArchaeoglobus; (b) adding an amount of the carbohydrate to the sample;(c) maintaining the sample under conditions suitable for fermentation ofat least a portion of the amount of the carbohydrate by thecarbohydrate-fermenting microorganisms, thereby producing anaerobicconditions in the sample; and (d) maintaining the anaerobic conditionsin the sample for a time that is sufficient for thenitroaromatic-degrading microorganisms to degrade the nitroaromaticcompound.